The present invention is directed to an improved method for identifying xe2x80x9corphan receptorsxe2x80x9d which involves a genetic selection for ligand-receptor interaction using a recombinant eukaryotic cell, preferably yeast, as a selection system.
Advances in molecular, cellular and viral biology have resulted in the identification of numerous transmembrane receptors. These advances have also made it possible to obtain transcripts and DNA encoding a range of proteins including putative transmembrane receptors. The identification of these receptors and putative receptors makes it possible to identify ligands that interact with these receptors permitting one to better understand the biology of those receptors and/or screen for compounds that modulate the effect of such receptors. However, an increasing problem is finding simple and accurate methods for identifying the specific ligands for each of these transmembrane receptors and putative transmembrane receptors. Those receptors for which a ligand has not yet been identified are referred to as xe2x80x9corphan receptorsxe2x80x9d. Such orphan receptors are becoming more numerous as more DNA sequences, including DNA sequences encoding putative receptors, become available.
Identifying the actual ligand that interacts with such receptors can be extremely important as many of these transmembrane receptors are associated with important cellular functions. For example, many transmembrane receptors have kinase activity and are growth factor receptors and some have been associated with malignant transformation of cells. For instance, growth factor independence in cancer cells has been correlated with overexpression of growth factor receptors such as erbB2 in breast cancer [19]. The overexpression of erbB2 has been shown to activate the ras/MAP kinase pathway and inhibition of the activation of this pathway has been shown to correlate with decreased cellular proliferation [3,9]. Potential links between tumor-associated overexpression of the erbB2 receptor and reduced survival of primary breast cancer patients with metastatic auxiliary lymph node involvement exists [1,2,23]. However, despite the considerable interest in erbB2, the specific ligand that interacts with it has not yet been identified making it an orphan receptor.
Being able to identify the actual ligand that interacts with a receptor such as the erbB2 receptor permits not only a better understanding of the complex physiological interactions involved, but facilitates the development of better drug assays. Thus, it would be desirable to have a better means for assaying and selecting ligands for these orphan receptors.
Another difficulty that currently exists in rational drug development is being able to identify when a ligand-receptor interaction occurs.
Compounds including small polypeptides that interact with not only orphan receptors, but other transmembrane receptors are currently screened by a wide variety of different assays. However, it would be desirable to develop new and simple assays to determine where an interaction is occurring not only to select compounds that modulate receptor activity, either positively or negatively, but to have a simple means to determine optimal peptide sequences that will, for example, fit into the ligand binding site. Identifying such compounds permits more efficient design of compounds that can be used in, for example, anti-cancer or anti-viral strategies. Thus, it would be useful to have a simple method for selecting only those cells where such ligand-receptor occurs.
We have now discovered a simple method for identifying ligands and ligand-receptor interactions involving transmembrane proteins that form dimers, preferably homodimers, for activation. Preferably, the transmembrane protein is a transmembrane receptor having protein kinase activity, such as a transmembrane tyrosine kinase receptor.
This method uses the unfolded protein response (UPR) pathway that is present in all eukaryotic cells, and conserved through evolution in organisms as divergent as mammals and yeast. The accumulation of unfolded proteins in the endoplasmic reticulum triggers a signal that is transmitted to the nucleus and results in increased transcription of chaperone proteins and enzymes that function to induce the correct protein folding. In the yeast, Saccharomyces cerevisiae, two of the essential components of the UPR pathway have been identified. In the presence of unfolded proteins, the transmembrane kinase IRE1p transmits a downstream signal that activates transcription of chaperone proteins and enzymes. This signal is manifested by the binding of nuclear factors to the unfolded response element (UPRE), a 22 bp upstream activating element having the sequence:
5xe2x80x2-GATCTGTCGACAGGAACTGGACAGCGTGTCGAAAAAGC-3xe2x80x2 (SEQ ID NO:1)
3xe2x80x2-ACAGCTGTCCTTGACCTGTCGCACAGCTTTTTCGAGCT-5xe2x80x2 (SEQ ID NO:2)
The UPRE is necessary and sufficient to activate transcription of a linked promoter in response to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) (Mori et al., EMBO J 11:2583-2593, 1992).
While the present method can be used in any eukaryotic cell that has a UPR pathway by using the IRE1/UPRE interaction or analog thereof, a preferred embodiment of the present invention involves using recombinant S. cerevisiae cell that contains a DNA segment encoding a IRE1/ERNI1 kinase domain fused to the extracellular domain of the receptor of interest, referred to as a chimeric receptor. Receptors of interest include, but are not necessarily limited to eukaryotic, viral, insect and mammalian receptors. Preferably, the receptor is a mammalian receptor. More preferably, the mammal is a human and the receptor is an orphan receptor. Like mammalian growth factors, IRE1 oligomerizes and is phosphorylated in trans in response to an accumulation of unfolded proteins in the endoplasmic reticulum (ER) [22]. When the appropriate ligand is secreted into the ER lumen, the chimeric receptor will oligomerize and activate the unfolded protein response (UPR) signaling pathway. Those cells where the pathway has been activated can readily be identified and selected by using a reporter system activated by the UPR signaling pathway. For example, one can use a responsive element such as the unfolded protein responsive element (UPRE) containing promoter fused to a marker gene such as LacZ [Cox, 1993]. Although the induction by the UPRE in an unfolded protein response is normally only two to four-fold because the protein is made at a high basal level, one can increase the level of induction by generating constructs containing multiple copies of the UPRE, and wherein the constitutive promoter elements of the marker genes are absent. Thus, the induction ratio would be much higher. The yeast colonies that have a UPR signal would turn blue on the Xgal indicator plates containing tunicamycin. Numerous other reporters can readily be used. For example, fusing the UPRE-containing promoter to the gene encoding the naturally green fluorescent protein (GFP) so that induction can be measured in living cells by fluorescence, thereby permitting the use of cell sorters. By this means, one can readily identify cells wherein a ligand has bound to a receptor, and induced the UPR signal. Thereafter, the cDNA encoding the putative ligand can be readily selected. In the instances where more than a single cDNA is selected, those cDNAs which actually encode a ligand which binds to the receptor can be readily resolved by transfecting individual cell lines containing the receptor with a vector containing one of the selected cDNAs encoding the putative ligand. One can then screen each of the transfected cell lines using standard methods (e.g., receptor binding assays) to identify those cells in which a ligand-receptor interaction occurs. The transfected cell can be a yeast cell of the present invention or any other cell that expresses the receptor protein.